MHCII with full peptide occupancy is a substrate for HLA-DM catalyzed peptide exchange

Abstract

Abstract The major histocompatibility complex class II (MHCII) antigen-processing pathway is critical for adaptive immunity as the mechanism through which ligands are generated for recognition by CD4 T cells. HLA-DM edits the repertoire of peptides that are presented to T cells by catalyzing multiple rounds of peptide exchange on MHCII molecules. The catalytic potency of DM varies such that peptide exchange is markedly enhanced on some peptide-MHC (pMHC) complexes and not others. This capacity to differentially edit complexes influences specificity and immunodominance in T cell responses. The catalytic mechanism and rules that govern DM susceptibility are not fully understood. Evidence suggests that partial occupancy at the N-terminus of bound peptides is critical for complex sensitivity to DM. We employed a fluorescence polarization assay to independently assess the effective affinity and catalytic turnover of DM, components of the catalytic mechanism, for pMHC complexes in real-time kinetic measurements of DM activity. We found that a complex with an N-terminal truncated form of the immunodominate HA peptide, resulting in the loss of three H-bonds mediated by the P-2 and P-1 residues, showed an expected decrease in complex stability and increase in affinity for DM binding, but an unexpected increase in the catalytic turnover when compared to the unmodified HA-MHC complex. This result was even more pronounced when a conserved bidentate H-bonding residue near the P1 anchor was disrupted by site-directed mutagenesis on an HA-MHC complex. Together, this implies that pMHC interactions near the N-terminal segment of a peptide may be intact in the functionally active DM-pMHC catalytic complex and provide insights into the DM catalytic mechanism.